Altered regulation of signaling in response to persistent cellular stress is associated with a number of complex human diseases, including cardiovascular, inflammatory and neurodegenerative disorders. Importantly, genetic studies have shown that silencing of the apoptosis signal-regulating kinase (ASK1) ameliorates many of the phenotypes manifest in mouse models of rheumatoid arthritis, cardiac hypertrophy and neurodegenerative disorders. Consequently, ASK1 has been highlighted as a therapeutic target where antagonists are anticipated to provide significant benefit in multiple disease states. Under the auspices of GM103957 our research team has recently completed a high throughput ASK1 screening campaign and identified a series of small molecule inhibitors from six different structural classes. We have narrowed the focus of our medicinal chemistry efforts to three scaffolds and the goal of this proposal is to develop an in vivo active ASK1 small molecule probe. In the studies of Aim 1 we will use an iterative medicinal chemistry approach to optimize the potency, selectivity and in vivo properties of ASK1 inhibitors aimed at peripheral targets. In addition, to develop compounds with suitable brain penetrant properties we will apply a multi-parameter optimization strategy focusing on physical and chemical properties that are hallmarks of the majority of current Food and Drug Administration (FDA)- approved CNS penetrant small molecule drugs. We will use structure based design to facilitate chemistry strategies and we will triage compounds through a battery of biochemical and cell-based assays as outlined in our research operating plan (ROP). As improved leads emerge, we will obtain enzyme selectivity for a select few, through full scale kinome (Reaction Biology) and receptor profiling (Riserca). For key leads we will also assess target engagement lifetime through biochemical and cell-based studies through measurement of ASK1 downstream substrate phosphorylation. Integrated into the ROP, we will also triage new compounds through a series of drug metabolism studies including assessment of in vitro microsomal stability, CYP450 inhibition, solubility, protein binding and P-glycoprotein efflux, with lead criteria integrated into the ROP for compound progression. In Aim 2, advanced leads for both peripheral and CNS exposures will be assessed in mice for adverse effects in acute dose range finding and 28-day toxicity studies. We will measure gross toxicity effect and vet scan analyses as well as TUNEL staining and histopathology of major organs. Finally, we will use inhibition of MKK6, JNK and p38 phosphorylation as in vivo reporters of ASK1 inhibition to quantify the maximum biological effective dose and through kinetic studies establish the PK/PD relationships of our lead in vivo probes. Our Multi-PI research team at the Scripps Florida campus of TSRI has both the expertise and infrastructure to perform these experiments, and we submit that the successful completion of our studies will identify a series of potent, selective and in vivo active molecular probes that will help define the role(s) that ASK1 plays in normal physiological processes as well as in disease states.